ABSTRACT
Purpose To investigate the relations of THEM4/Akt expression and extracellular matrix deposit in kidney of diabetic mice. Methods Diabetic mice models were successfully established by intraperitoneally injected STZ. Both normal control mice and diabetic mice were raised for 8 week until they were sacrificed. Western blot, immunohistochemistry and realtime PCR were used to detect the expression of THEM4, phospho-Akt (Ser 473), TGF-β1, a-SMA, Col Ш, FN protein and THEM4 mRNA in the kidneys of normal mice and diabetic mice. Results Compared to normal control mice, THEM4 expression decreased by 37.7％ followed by 3.66, 1.29 2.33, 1.99 and 2.82 times increased of phospho-Akt (Ser473), TGF-β1, a-SMA, Col Ш and FN in kidney of diabetes mellitus. Extracellular matrix accumulation was found in renal interstitial region of diabetic mice. Conclusion The decreased THEM4 might cause extracellular matrix deposit in kidney of diabetic mice by upregulating the phosphorylation of Akt and TGF-β1, α-SMA expression in diabetic mice.
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Aim To investigate the effect of thioester-ase superfamily member 4(THEM4)expression on col-lagen secretion in human renal proximal tubular epithe-lial cells (HKC)treated with high glucose. Methods In order to examine the direct effect of THEM4 ex-pression vector on PI3K/ Akt pathway and collagen se-cretion,pYr-ads-4-THEM4 expression vector was con-structed and transfected into the HKC with lipo-fectamine 2000 in vitro. HKC cells were randomly di-vided into four groups:normal glucose group (Con-trol),high glucose group (HG),high glucose plus pYr-ads-4-THEM4 vector group (HG + THEM4 vec-tor) and high glucose plus pYr-adshuttle-4 vector group (HG + V vector). After 48 h with HG stimula-tion,the cells were collected for extraction of protein and phospho-Akt (Ser 473),THEM4,TGF-β1 andα-SMA protein expression were examined by Western blot and immunofluorescence staining respectively. Col Ⅰ and Col Ⅲ were detected using the competitive sandwich ELISA kit according to the manufacturer's instructions. Results High glucose inhibited THEM4 expression,and induced increased phospho-Akt (Ser 473),TGF-β1,α-SMA and secreted ColⅠand secre-ted Col Ⅲ in HKC cells. Up-regulation of THEM4 re-versed high glucose-induced decreased THEM4,in-creased phospho-Akt (Ser 473),TGF-β1,α-SMA, secreted Col Ⅰ and secreted Col Ⅲ in HKC cells. Conclusion The up-regulation of THEM4 may de-crease Col Ⅰ and Col Ⅲ secretion by inhibiting the phosphorylation of Akt and down-regulating the expres-sion of TGF-β1 and α-SMA in high glucose-induced HKC cells.
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Purpose To investigate the expression of XBP-1s and ADRP in kidney of diabetic rats. Methods Diabetic rat models were successfully established by intraperitoneal injection of STZ. After two months rats were sacrificed and XBP-1s and ADRP were de-tected by immunohistochemistry and Western blot. Results XBP-1s and ADRP were located in renal tubular cells and increased by a-bout 2. 017 times and 1. 544 times in comparison with normal control rats (P<0. 05). Moreover, it was shown that high expression of XBP-1s was commonly accompanied with increased ADRP by Pearson correlation analysis and the correlation coefficient was 0. 723 (P<0. 05). Conclusion The increased XBP-1s may cause the up-regulation of ADRP in the kidney of diabetic rats.
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Aim Todeterminetherelationshipbe-tween serum TNF-α and renal abnormal lipid metabo-lismindiabeticmice.Methods CD1micewerein-jected intraperitoneally with STZ (150 mg·kg-1 ) and the type 1 diabetic mice were determined with high fasting blood glucose ( >16. 7 mmol · L-1 ) 72 hours after injection. After fed for one month, normal control mice and diabetic mice were sacrificed. The serum TNF-α level was detected by the method of ELISA. Immunohistochemistry and Western blot were used to explore the expression of SREBP-1 and ADRP. Re-sults TheresultsofELISAshowedthatserumTNF-αwas increased by 7 . 73 times in diabetic mice compared with normal mice. SREBP-1 and ADRP expressions were located in renal tubular cells. Again, it was con-firmed by Western blot that SREBP-1 precursor seg-ment, SREBP-1 mature segment and ADRP were re-spectively enhanced by 2. 31 times, 1. 74 times and 1. 72 times in diabetic mice in comparison with normal control mice . In addition , the data analysis revealed a positive correlation ( correlation coefficient 0. 914 ) be-tween serum TNF-αand renal ADRP expression. Con-clusion TheincreasedserumTNF-αmaybeoneof factors involved in renal lipid accumulation of diabe-tes.
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Objective To investigate the effects of metabolically correlated factors on pig renal tubule in cellular level by observing effects of glucose,insulin and uric acid on activity of haemachrome oxygenase in renal tubular epithelial cells.Methods Pig near renal tubular cell line(LLC-PK1) was selected as experimental object.After stimulating cells respectively with glucose(5,10,22,33mmol/L),uric acid(0,0.1,0.2,0.4mmol/L);and insulin(0,10~(-9),0~(-8),10~(-7)mol/L) in different concentrations for 48 hours,the activity of HO-1 within the cells was determined.Results In uric acid group the activity of HO-1 was significantly increased in a concentration-dependent manner,while in glucose and insulin groups,the activity of HO-1 did not change.Conclusion After LLC-PK1 was stimulated by uric acid of different concentrations,HO-1 was induced evidently,which indicates that uric acid might affect oxidative stress in renal tubule cell line.
ABSTRACT
Thickening of tubular basement membrane and progressive tubulointerstitial fibrosis has been reported as important components of diabetic nephropathy, In order to investigate the mechanisms of tubulinterstitial changes in diabetic nephropathy, we evaluated the effects of a high concentration of glucose(25mM; 450mg/dL) on glucose transporter GLUT1 level, fibronectin production and tissue inhibitors of metalloproteinases (TIMP)-1 concentration in renal tubular(LLC-PK1) cells. As the effect of high glucose-induced alteration in LLC-PK1 cells, the expression of facilitative glueose transporter, GLUT1 was decreased after longer than 24-hours exposure to 25mM glucose, compared to control(5.6mM). The administration of protein kinase C (PKC) inhibitor GF109203X(10 microM) did not show significant effect on high glucose-induced decrease of GLUT1 level. On western blot analysis of fibronectin production, The exposure of LLC-PK cells to 25mM glucose for 48 hours significantly increasc4 fibro- nectin production, dose-dependently. The addition of GF102903X at the concentration of 10pM induced the significant increase of fibronectin level in LLC-PK1cells under glucose-free condition, whereas there was no significant effect on the high glucose-induced increase of fibronectin production. The addition of anti-TGF-beta antibody at 30 microgram/mL partly inhibited the high glucose-induced increase of fibronectin production. Concerning the changes of tissue inhibitor of metallo-proteinase(TIMP)-1 levels in the presence of high glucose, the exposure to high glucose for 24 and 43 hours increased TIMP-1 levels in culture supernatant of LLC-PK1 cells, dose-dependently. The TIMP-1 levels of 48-hour exposure to 15 and 25mM glucose were also significantly higher than those of 24-hourexposure. The treatment with 10 microM GF102903X or 30 microgram/mL anti-TGF-Beta antibody had no significant effects on TIMP-1 levels measured under the high glucose culture condition. In conclusion, the expression of facilitative glucose transporter, GLUT1 is inhibited and the production of fibronectin is increased in renal tubular cells cultured in the presence of high concentration of glucose, which is partly mediated by TGF-beta. The TIMP-1 level is also increased under high glucose culture condition. The enhanced productions of fibronectin and TIMP-1 of renal tubular cells under high glucose concentration may contribute to tubulointerstitial fibrosis that occurs in diabetic nephropathy.
Subject(s)
Animals , Basement Membrane , Blotting, Western , Diabetic Nephropathies , Epithelial Cells , Extracellular Matrix , Fibronectins , Fibrosis , Glucose Transport Proteins, Facilitative , Glucose , LLC-PK1 Cells , Metalloproteases , Protein Kinase C , Swine , Tissue Inhibitor of Metalloproteinase-1 , Transforming Growth Factor betaABSTRACT
Objective To study the cytopathogenic effect of epidemic hemorrhagic fever with renal syndrome virus (HFRSV) on renal tubular cells(RTC). Methods Human fetal renal tubular cells (HFRTC) were cultured in vitro. HFRTC infected or not infected by HFRSV were observed by using trypan-blue stain and transmission electron microscopy(TEM). Viral-mRNA was detected by in situ molecular hybridization. Results (1) HFRSV could directly infected HFRTC: (2)The death rate of HFRTC in the infection group was significantly higher than that in the control grou 1 to 4 weeks after infection; (3) Injuries of cell membrane and cell organs after infection with HFRSV were significantly earlier and more severe as compared to control by means of TEM. Conclusion HFRSV can directly damage renal tubular cells (RTC ), which contributes to the pathogenesis of hemorrhagic fever with renal syndrome (HFRS).